Characteristics Of Interstitial Cells Of Cajal And Associated Nerves In Oviduct Of Chicken

The oviduct is the important germinative organ that promises the transportation and maturation of the eggs in a well-regulated and controlled manner. It is a factory where the eggs/embryos-the future animals are produced. The increasing demand of the modern world for the proteins of the animal origin has made it crucial to develop strategies for the improved production from livestock and poultry species. The poultry species provide a good source of the proteins of animal origin; in the form of eggs and short maturity age of the broilers. The animals constitute the unique source of the milk production and meat production. The healthy female animals are the key to supply the needs of the developing world. Understanding of the normal functioning of the female reproductive system is thus of the great importance to design the future animals that are healthy and more productive. In this context, the knowledge on the advancement in the structure of the oviduct is fundamental to achieving such goals because this organ is concerned to the transport of gametes, fertilization, and early embryonic development. Recent studies have established the role of interstitial cells of Cajal (ICC) as the founding regulators of neuromuscular contractile axis. The information on the localization of these cells in the oviducts of chicken is widely lacking. We have demonstrated the presence of ICC in the chicken oviduct also characterized these cells on the basis of their ultrastructure. The molecular evidence of the regulation of ICC at gene level has also been investigated. Furthermore, we have elaborated the localization of the nitrergic and cholinergic nerves in the oviduct of chicken. The relative localization studies on the ICC (Chapter 3) and neurons in this study clearly demonstrate that ICCs preferably reside at the places where the nitrergic and cholinergic nerves were localized (Chapter 5 & 6). We also have studied the ICC and nitrergic and cholinergic nerve populations in the goat oviductal isthmus. The differences are obvious, although frequent overlaps have also been observed at the structural level among the two species.Thus, the present study has broadened our knowledge and meanwhile has given a new perspective on the oviductal motility in chicken and goat. Furthermore, it provides a ground work at the structural level to study the important processes of the fertilization in the livestock species. Further studies will be required to observe these phenomenons on the functional and genetic/molecular grounds.Experiment I (Chapter 3) Identification and Ultrastructural Characterization of Interstitial Cells of Cajal (ICC) in Chicken OviductThe interstitial cells of Cajal (ICC) are widely believed to be neuroeffector cells of smooth muscle activity in all tubular organs in mammals, including the oviduct. The avian oviduct involves the secretion, sheathing, and transportation of a large-sized egg, but there is no information available on ICC in this special organ. We have demonstrated the presence of ICC in different segments throughout the oviduct in the laying hen, and provided details on their ultrastructure by transmission electron microscopy technique, for the first time. The observed ICC appeared bipolar and multipolar cells of different shapes, with varying nuclear morphologies, a thin rim of electron-dense cytoplasm, and an infrequent basal lamina. They showed moniliform primary processes with one or 2 secondary or terminal processes. We found ICC near smooth muscle cells, nerve fibers, and the epithelia, where they make specialized contacts in the form of close membrane associations or gap-like junctions and peg-and-socket-like junctions. Intricate labyrinthine-type networking contacts were also present in ICC processes. Moreover, we report for the first time, that ICC in avian oviduct make interdigitating contacts with the epithelium. Cytoplasmic organelles identified in ICC include numerous well-developed mitochondria, abundant rough endoplasmic reticulum, and dispersed intermediate filaments. Many caveolae and vesicles were also present. Golgi bodies and centrioles were rare. Fibroblasts, on the other hand, were distinct cells with larger Cytoplasmic area, more rough endoplasmic reticulum, and less mitochondrial content. No basal lamina, intermediate filaments, or caveolae were present in fibroblasts. Their processes were shorter and showed no contacts with smooth muscle cells or nerves. We conclude that these ICC might also have a key role in the regulatory mechanisms of motility and transportation in the hen oviduct, as already proved in mammalian oviduct. Such role of ICC might also be responsible for the function of the muscular infundibulum, where the fertilization takes place, and that moves to surround the released ovum, failure of which results in the internal laying.Experiment II (Chapter 4) The Expression of c-Kit mRNA in Chicken Oviductc-Kit is an oncogene, a member of tyrosine kinase family, it is believed as a specific marker of the interstitial cells of Cajal (ICC). Using the quantitative real-time polymerase chain reaction (RT-qPCR), we have demonstrated the mRNA expression of c-Kit gene in the five segments of the chicken oviduct, the infundibulum, magnum, isthmus, uterus (shell gland), and vagina. The TEM observations showed high number of ICC in the muscularis of the chicken oviduct; therefore, we used the muscular layer from each segment to determine the c-Kit expression. The c-Kit mRNA was expressed throughout the oviduct, with regional variations in the levels of expression. The highest level of Kit mRNA expressed was found in the magnum segment of the oviduct. The next segment showing relatively high mRNA expression was the infundibulum, followed by the uterus. Moderate levels of expression were seen in the vagina while the isthmic region showed low level of expression of the c-Kit gene. The regional variation in the relative expression of the c-Kit gene may have some functional grounds in the control of motility of the oviduct.Experiment Ⅲ (Chapter 5) Histochemical Localization of Nitric Oxide Synthase Positive Nerves in Chicken OviductNitric oxide (NO) is an important inhibitory neurotransmitter, synthesized by the enzyme nitric oxide synthase (NOS) locally in many tissues. The histochemical technique using nicotinamide adenine dinucleotide-diaphorase/nitric oxide synthase (NADPH-d/NOS) is often employed to determine the presence of this enzyme in the neuronal as well as other cell populations. We followed this principle to determine the NOS activity in the five segments, viz. infundibulum, magnum, isthmus, uterus (shell gland) and vagina of chicken oviduct. The reaction was seen in the nervous structures, the vascular endothelium and epithelium. The observed positive neuronal somata appeared unipolar, to bipolar or multipolar with spindle, rounded, or oval shapes, with the small nucleus displaced to one pole of the soma. The positive nerve structures were very scant in the infundibulum but the population of positive nervous structures and fibers increased in the posterior segments. The somata were distributed in the lamina propria, the intermuscular tissue and near the blood vessels, in the muscularis, and in the subserosal connective tissue in variable numbers. The cells were usually seen as single soma, but they were also arranged in pairs or groups of 3-4 cells, the number varied in different segments. The fibers ran in the muscularis, in the lamina propria and the intermuscular region. The number of positive soma was lowest in the infundibulum, the magnum showed an increased number of somata, but reduced again in the isthmus. The uterus and vagina showed more developed neuronal cell bodies, with the vagina showing the highest number of somata. Both, in the uterus and vagina the somata showed more elaborated pattern of fibers running in various directions at various levels in the wall. Since the somata or fibers were commonly found near the blood vessels, it may be concluded that the nitrergic neuronal population constitutes important regulatory neuronal molecules of the oviduct blood flow, in addition to their role in controlling the oviductal smooth muscle contraction.Experiment IV (Chapter 6) Histochemical Localization of Acetylcholinesterase Positive Nerves in Chicken OviductThe motility of the oviduct is not just the cellular interplay, but there are complex underlying phenomenon brought about by the specific molecules, the neurotransmitters that drive this activity in a highly organized manner. Acetylcholine is an important excitatory neurotransmitter that causes the contraction of smooth muscle. We have demonstrated the localization of acetylcholine in oviductal tissues in the hen oviduct. We used the acetylcholine iodide as a substrate to label the acetylcholinesterase positive neuronal structures in hen oviduct by histochemical technique. The cells were arranged as single cells, and in clusters of two to three cells. The cytoplasm and the processes of positive neurons showed strong reaction, the nucleus showed no staining. The nucleus was eccentric lying at one pole of the cell. The morphology of the stained neurons was rounded and oval in shape. Similar features were seen in the whole mounts. The polar characteristic appeared to be unipolar, bipolar, and sometimes multipolar cells. Varicose nerve fibers were also observed. The regional distribution of the cholinergic nerve structures was most commonly observed in the muscularis, where the cells were either seen as solitary cells or in groups or clusters. The cells in the muscular layer were sometimes seen as tracts of cholinergic nerves whose processes run along the muscular layer and sometimes make a bifurcation and enter the lamina propria of the mucosal folds. The positive staining cholinergic neuronal fibers traversed along the length of the lamina propria of the mucosal folds and also innervated the secondary and tertiary mucosal folds. The cholinesterase positive nerve structures also preferably targeted the blood vessels. The specific neuronal populations appeared to be higher in the proximal oviduct; while the lower population of positive nerve structures was seen in the isthmus and vagina of the avian oviduct. Our results suggest that acetylcholine might be one of the key molecular coordinators of the oviduct motility in laying female chicken.Experiment V (Chapter 7) Observations on the Localization of Interstitial Cells of Cajal, Nitric Oxide Synthase and Acetylcholinesterase Positive nerves in Goat Oviductal Isthmus and Comparison with the Chicken OviductIn the goat oviductal isthmus, the ICC showed spindle shaped bipolar morphologies with a large nuclear to cytoplasmic content ratio, numerous mitochondria, rough endoplasmic reticulum and many caveolae. They were frequently found near the nerve fibers and the blood vessels. No specialized contacts could be observed between the ICC and the muscles or nerves, except the labyrinthic contacts between the ICC processes. We also determined the nicotinamide adenine dinucleotide diaphorase (NADPH-d) or nitric oxide synthase (NOS), and acetylcholinesterase (AChE) activities in the goat oviduct. The NOS positive structures were distributed at all levels in the goat isthmus, and followed the pattern more or less similar to that seen in the avian oviduct as far as the location is concerned. The reaction was variably seen throughout the different layers in the isthmus; the epithelium, the lamina propria, the circular muscle layer and the thin outer longitudinal muscle under the serosa; and near the blood vessels. The somata were mostly seen in groups of 2 or more cell bodies; however single cells were not uncommon. The AChE activity of the oviductal isthmus revealed the positive cell bodies found in the external muscular layer, and there was a relative reduction of cell bodies together with an increase in the fibers. The lamina propria was supplied by the fibers extending from the inner circular muscle layer. The cholinergic fibers also innervated the blood vessels; while the epithelium of the isthmus did not appear to show positive reaction. The identification of ICC, and histochemical staining in the goat oviductal isthmus, presumably, is reported for the first time. Since the most preferable localization of the ICC, nitrergic and cholinergic somata or fibers was near the blood vessels, it may be concluded that the ICC, and the nitrergic and cholinergic neuronal populations constitute unique regulatory cells and molecules regulating the oviduct blood flow in the goat oviductal isthmus, in addition to deriving its smooth muscular activity.